EP0274911B1 - Diagnostisches Hilfsmittel, seine Herstellung und seine Verwendung - Google Patents

Diagnostisches Hilfsmittel, seine Herstellung und seine Verwendung Download PDF

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Publication number
EP0274911B1
EP0274911B1 EP87311509A EP87311509A EP0274911B1 EP 0274911 B1 EP0274911 B1 EP 0274911B1 EP 87311509 A EP87311509 A EP 87311509A EP 87311509 A EP87311509 A EP 87311509A EP 0274911 B1 EP0274911 B1 EP 0274911B1
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EP
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Prior art keywords
membrane
diagnostic device
test field
liquid
indicator medium
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French (fr)
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EP0274911A1 (de
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Peter John Degen
Thomas C. Gsell
Vlado I. Matkovich
Jerold Martin
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Pall Corp
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Pall Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements

Definitions

  • This invention relates to a device for performing clinical diagnostic tests and methods for carrying out these tests. Specifically, it relates to a device and methods for detecting and quantifying the presence of biologically-active materials. More specifically, this relates to a device comprising a microporous membrane, on which detection reagents are immobilized, which membrane is bonded to an inert support, enabling convenient handling of the device as it is inserted into and removed from materials to be analyzed. The invention also relates to a method for making diagnostic devices.
  • test kits whereby the presence of simple chemicals, such as sugar, or even more complex materials such as enzymes, can be estimated in biological fluids, e.g., blood or urine.
  • biological fluids e.g., blood or urine.
  • Such kits consist of absorbent materials which are impregnated with a reagent or a combination of reagents which produce a change in color in the presence of certain biological substances.
  • the matrix is first immersed in the fluid in order to contact the reagents with the substance in the fluid of interest. Then, the matrix is removed from the liquid and a color change is noted. In many cases, the matrix must be washed or developed with a series of reagents to cause a visible reaction. The visible change indicates the presence of the biological chemical sought.
  • Such test kits are used, e.g., to detect glucose in urine and to detect phenylketonuria (phenylpyruvic oligophrenia or PKU).
  • test kits are useful because they often require only small amounts of the fluid to be tested (specimen), little preparation of the specimen, and the analysis is usually rapid. However, the results of such tests are not quantitative, except in certain cases where the substance to be analyzed is present in relatively large amounts, for example, glucose in urine.
  • small amounts of a toxin can be detected by the following method.
  • the material to be analyzed can be exposed to a matrix on which an antibody to the toxin is immobilized.
  • An antibody is a protein produced by living cells of an animal which has a specific affinity for another (usually foreign) biological substance with which the animal may come in contact. Any of this toxin in the specimen will then form an affinity complex with the antibody on the matrix. Because antibodies are specific for the substance with which they complex, only the toxin whose antibody is immobilized on the matrix will bind to it. No other toxins or other materials in the specimen will form a complex with the antibody on the matrix. Therefore, the specimen does not need to be purified or converted to a more convenient form prior to analysis.
  • the matrix can then be exposed to a solution containing another antibody to the toxin.
  • This antibody must have first been modified with a chemical marker or "label" which enables it to be easily detected.
  • the label could be a radioactive atom, a fluorescent dye, or an enzyme which can react with other chemicals to produce a visible color.
  • the labeled antibody will bind to the matrix only in the amount that the toxin was bound to the matrix in the previous step because the labeled antibody binds only to the toxin.
  • the labeled antibody can be easily detected and quantified. Because it is present in the same amount that the toxin is present, the toxin is also quantified by this method.
  • This method Several variations of this method are known, all of which are based on the abilities of antibodies to form specific complexes with certain other biological substances (referred to as "antigens"). Such tests are able to detect and diagnose such diseases as serum hepatitis.
  • the technology now exists to produce large quantities of antibodies which are specific to antigens of many types. Therefore, it should be possible to create diagnostic test equipment to detect a great many clinically important substances.
  • the diagnostic device comprises an elongated support member having secured to at least a portion thereof a liquophilic, microporous, synthetic, polymeric membrane indicator medium or test strip, the membrane having a surface area of from 2 to 12 m2/gram, a pore rating of from 0.04 to 10 micrometers with a pore size distribution of no greater than ⁇ 15% and the ability to immobilize the reagent thereon in a reproducible manner.
  • the polymeric membrane is an alcohol-insoluble polyamide membrane or a polyvinylidene difluoride membrane.
  • the invention is also directed to a diagnostic method for testing a liquid to quantitatively detect biologically-active material in the liquid comprising contacting a liquophilic, microporous, synthetic, polymeric membrane indicator medium or test field secured to at least a portion of an elongated support member and having a reagent immobilized thereon with the liquid containing the biologically-active material to be analyzed for, the membrane having a surface area of from 2 to 12 m2/gram, a pore rating of from 0.04 to 10 micrometers with a pore size distribution of no greater than ⁇ 15% and the ability to immobilize the reagent thereon in a reproducible manner.
  • the invention is further directed to a method of preparing a diagnostic device for biologically-active materials comprising treating an indicator medium or test field material to immobilize one or more test reagents thereon and thereafter preparing a diagnostic device from a portion of the treated indicator medium or test field material.
  • the tests based on antibody-antigen interaction and tests based on identification of genetic material each involve the reaction of two reagents.
  • the reaction takes place between antibodies and antigens and, in the second case, between DNA and gene-specific chemicals referred to as "DNA probes".
  • DNA probes DNA probes
  • one component of the reactive system - an antibody, an antigen, or a DNA probe - is a small molecule that can penetrate the pore structure of most matrices which can be used as supports.
  • the second component - an antigen or genetic matter - can be present in forms which vary in size from molecular dimensions to whole cells several microns in diameter.
  • the materials selected as the porous support matrix i.e., the indicator medium or test field, must meet several criteria. These criteria are: (1) a predetermined, high total available surface area, (2) a carefully defined and controlled pore size and pore size distribution, (3) liquophilicity, preferably hydrophilicity, and (4) the ability to immobilize, in a reproducible manner, the selected reagent.
  • the pore size of the support must be large enough for the largest reagents - antigens or genetic matter - to penetrate the support. It is also important to maximize the surface area of the indicator medium to enable immobilization of the largest amount of reagent as possible to enhance sensitivity.
  • surface or “surface area” refers not only to the gross surface(s) of the medium, but also to the surfaces of the micropores of the medium, i.e., the interior surfaces which are contacted by fluid during use. Available surface area decreases as pore size increases.
  • the optimum pore size for the support should be the smallest pore size which will permit complete penetration of the largest reactant. This size will vary from submicron dimensions when the largest reactant is a virus, an enzyme, or an antibody to several microns when the largest reactant is a whole cell or a cell fragment. The ease and completeness of penetration is also effected by the wetting characteristics of the indicator medium as discussed below.
  • the indicator medium or test field of a diagnostic device should, therefore, be capable of having immobilized on its surface, in a uniform manner, a fixed amount of reagent per unit volume of indicator medium or test field (membrane).
  • a "unit volume" of the medium is meant the volume occupied by a section of the medium (membrane), i.e., a given planar membrane area and thickness.
  • a voids volume e.g., 70%, which is available for penetration of the reagent - containing liquid medium.
  • the surface area of the indicator medium or test field of the unit volume, as noted above, must be high to immobilize the largest amount of reagent as possible to enhance sensitivity. Further, to provide quantitative results, the surface area of the membrane in that unit volume, as well as the balance of the indicator medium or test field, must be uniformly distributed to insure uniform immobilization of the reagent.
  • the total amount of the reagent immobilized will be determined by the binding capacity per unit volume of the indicator medium or test field and by its total volume.
  • the reproducibility of the test will depend on the uniformity of the medium. Therefore, to provide a diagnostic device with quantitative results, high sensitivity, and good reproducibility, it is necessary to use a fixed amount of reactant immobilized in a uniform manner on a fixed volume of uniform medium with high binding capacity, whose pore size has been determined to meet the requirements set forth above, i.e., the pore size must be large enough to allow access of the largest reagent while providing adequate surface area, bearing in mind that when pore size increases, surface area decreases.
  • Materials found to be useful as the indicator medium or test field in the diagnostic devices in accordance with this invention are liquophilic, microporous membranes as described in more detail below.
  • the indicator medium or test field must not react adversely with substances found in either the samples, reagents, or solvents employed in the analyses. Further, the membranes used should be essentially free of extractables which would adversely affect the particular test being carried out. To ensure good penetration and uniform distribution of the test reagents, the membrane must be liquophilic, i.e., spontaneously wettable by the test fluids. In addition, for the reasons discussed above, the indicator medium or test field must have a high surface area coupled with a pore size which will allow penetration of the largest test reagent or reactant.
  • the K L is essentially equivalent to bubble point, foam-all-over point, and other similar techniques familiar to those skilled in the art.
  • Pore rating is related to K L by the formula: where D is the pore rating in micrometers, K L is the measured K L in psi, and C is a constant, which has a value of about 10 when using water as the wetting fluid for K L measurement. This relation is described in Publication NM 900a, dated September 1980, available from Pall Corporation.
  • the pore size distribution of the membranes used as the indicator medium or test field typically have pore size distributions of no more than ⁇ 15% of the pore rating determined in the manner described above, preferably of no more than ⁇ 10%.
  • Membranes which are suitable for use as the indicator medium or test field typically have voids volumes in the range of 60 to 90%, preferably in the range of 70 to 90%.
  • Preferred materials are hydrophilic in nature and are, therefore, easily water-wettable and tend to freely pass aqueous solutions.
  • Liquophilic, microporous membranes useful in accordance with the present invention include alcohol-insoluble polyamide membranes and polyvinylidene difluoride membranes which have been rendered liquophilic by appropriate treatment, such as by surface modification.
  • alcohol-insoluble polyamide membranes included within the term alcohol-insoluble polyamide membranes are modified polyamide membranes, containing as a major component thereof, an alcohol-insoluble polyamide.
  • Liquophilicity refers to the spontaneous wettability of the membrane by the liquid(s) with which it is contacted.
  • the wettability or liquophilicity of a structure is a function of that structure's critical surface energy and the surface tension of the applied liquid. If the critical surface energy is at least as high as the surface tension of the liquid, then the liquid will spontaneously wet the structure.
  • a microporous membrane having a critical surface energy of 0.72 N/M (72 dynes/cm) or higher will be wetted by water which has a surface tension of 0.72 N/M (72 dynes/cm), i.e., the membrane is hydrophilic.
  • Suitable membranes must be capable of being treated with and retaining or immobilizing the reagent used to perform a specified test or reaction with the substance being analyzed for in a sample. As noted above, to obtain quantitative results, the reagent must be immobilized on the surface of the membrane in a uniform manner, in a fixed amount, and in a reproducible manner.
  • the necessity for uniform immobilization and high sensitivity dictates the use of a uniform, homogeneous membrane and precludes the use of a fibrous/resin-based system as the indicator medium due to (1) the non-homogeneity of the system, (2) the inherent low surface area of the fibers, and (3) the non-discriminating pore sizes of such fiber-based systems with widely varying pore sizes resulting from random crossing of fibers.
  • the reactant which may be of ionic, molecular, or macromolecular nature, may be immobilized on the reaction layer by strong physical forces or by being bonded in some manner, such as by covalent chemical coupling, to the surface of the membrane layer.
  • wettability or liquophilicity is a requisite of the membranes used as the indicator medium or test field in accordance with the present invention to (1) ensure complete and uniform penetration of the reagent-containing liquid and, hence, full use of the membrane and uniform immobilization of the reagent on the surface of the membrane, as well as (2) complete and uniform penetration of the liquid containing the analyte, i.e., the substance being analyzed for.
  • Preferred membranes used as the indicator medium or test field in accordance with the present invention are intrinsically hydrophilic or water-wettable.
  • the unique high-binding capacity, uniformity, controlled pore size, and high surface area of the hydrophilic, microporous nylon 66 membranes described in U.S. Patent 4,340,479 and available commercially from Pall Corporation under the trademark BIODYNE® A make them particularly useful as the indicator medium or test field.
  • the hydrophilic, microporous, polyamide filter membranes disclosed in U.S. Patent 4,340,479 are membranes prepared from alcohol-insoluble polyamide resins having a methylene to amide ratio in the range of 5:1 to 7:1.
  • Membranes of this group include copolymers of hexamethylene diamine and adipic acid (nylon 66), copolymers of hexamethylene diamine and sebacic acid (nylon 610), homopolymers of poly- ⁇ -caprolactam (nylon 6), and copolymers of hexamethylene diamine and azelaic acid (nylon 69).
  • POSIDYNE® is a hydrophilic, microporous, skinless nylon 66 membrane with the surface properties controlled by quaternary ammonium groups of a polyamine-epichlorohydrin polymer or resin cocast with the nylon 66.
  • IMMUNODYNETM is a modified CARBOXYDYNE® membrane, also available from Pall Corporation.
  • CARBOXYDYNE® is a hydrophilic, microporous, skinless nylon 66 membrane with controlled surface properties formed by the cocasting process described in U.S. Patent 4,707,266, as discussed below, specifically by cocasting nylon 66 and a polymer containing an abundance of carboxyl groups to form a membrane having controlled surface properties characterized by carboxyl functional groups at its surfaces.
  • IMMUODYNETM membranes may be prepared from CARBOXYDYNE® membranes by treating them with trichloro-s-triazine in the manner described in U.S. Patent 4,693,985, discussed below.
  • Polyvinylidene difluoride membranes are not inherently water-wettable, but can be rendered such by an appropriate surface treatment.
  • Microporous, polyvinylidene difluoride membranes which have been treated to render them hydrophilic are commercially available, e.g., from Pall Corporation under the trademark FLUORODYNETM and from Millipore Corporation as hydrophilic DURAPORE®
  • wettability or liquophilicity is a function of the critical surface energy of the structure and the surface tension of the liquid. Wettability may also be expressed in terms of intrusion pressure required for liquid to penetrate into the pores of the membrane.
  • membrane materials which are particularly preferred have intrusion pressures of, or close to, zero.
  • membranes useful as the indicator medium have large surface areas, typically from 2 to 12 m2/gram or even higher. This characteristic permits a greater amount or higher concentration of reactant to be immobilized in the indicator medium. Accordingly, higher sensitivities may be achieved using the diagnostic device in accordance with the present invention.
  • polyamides preferred for use in accordance with the present invention include alcohol-insoluble nylons of the type described in U.S. Patent 4,340,479.
  • a membrane material of this description which is particularly useful for the present invention is a microporous, hydrophilic nylon membrane (nylon 66) commercially available from Pall Corporation under the trademark BIODYNE®.
  • hydrophilic, microporous, skinless, alcohol-insoluble polyamide membranes with controlled surface properties are also included among the preferred polyamide membranes for the present invention.
  • hydrophilic, microporous, substantially alcohol-insoluble polyamide membranes with controlled surface properties are formed by cocasting an alcohol-insoluble polyamide resin with a water-soluble, membrane-surface-modifying polymer having functional polar groups.
  • the polyamide membranes used as the indicator medium or test field in the present invention having controlled surface properties are also skinless; that is, they are characterized by through pores extending from surface-to-surface which are of substantially uniform size and shape. If desired, however, materials having tapered through pores, i.e., pores which are larger at one surface of the sheet, narrowing as they approach the opposite surface of the sheet, may be used.
  • the surface-modifying polymers used to prepare the polyamide membranes with controlled surface properties comprise polymers which contain substantial proportions of chemically functional groups, such as hydroxyl, carboxyl, amine, and imine groups.
  • the membranes include, at their surfaces, high concentrations of functional groups such as hydroxyl, carboxyl, amine, imine, or a combination of any of the above groups which do not react with one another.
  • polyamide membranes having controlled surface properties have higher concentrations of carboxyl or imine groups at their surfaces than the other preferred alcohol-insoluble, microporous, hydrophilic, skinless polyamide membranes described above which do not have controlled surface properties, i.e., those which are formed from the preferred polyamide resin but are not cocast with surface-modifying polymer.
  • the membranes used may be treated by any method known to one of skill in the art to deposit and/or bind reagents thereto.
  • the reagent may be of an ionic, molecular, or macromolecular nature.
  • the reagent When used as a diagnostic tool to provide a visible change, the reagent may be one or a combination of substances which is initially colorless and which, upon reaction with a suitable material, provides an optically measurable response.
  • suitable labels such as the formation between the deposited reagent and the material for which testing is being conducted of a complex or compound which is appropriately labeled by any known technique, such as enzymatic/substrate labels or the like.
  • Treatment of the indicator medium or test field (membrane) of the diagnostic device with a suitable reagent(s) may be performed at the time at which diagnostic tests are to be performed.
  • the test reagent(s) may be added immediately preceding contacting of the test device with the liquid containing the sample to be analyzed for, e.g., by "spotting" the reagent(s) on the membrane secured to the support member.
  • the present invention is expected to have greatest application to, and a preferred embodiment includes, diagnostic devices prepared by treating indicator medium or test field material with one or more test reagents to immobilize one or more test reagents thereon, and thereafter preparing the individual diagnostic devices from a portion of the treated material.
  • a ribbon or continuous strip of a membrane may be treated with the reagent(s) and then that membrane applied to the material to be used as the support member following which individual diagnostic devices are manufactured.
  • This will be more clear be reference to Example 1 below.
  • Other variants include (1) placing a treated ribbon of membrane on top of a second wider untreated ribbon of the same membrane to provide a controlled background medium prior to manufacturing of the individual diagnostic devices and (2) applying a band of the reagent(s) to a ribbon of membrane to provide the controlled background within a single piece of membrane.
  • This patent describes a method for immobilizing a wide range of biologically active substances as acceptor molecules on active membranes.
  • the acceptor-bound membranes described in the patent are capable of immobilizing and binding a wide variety of biologically-active compounds, specifically ligands, to the acceptor molecules.
  • Using such reaction layers or membranes permits the testing of bodily fluids, such as blood, serum, plasma, urine, salivas, and the like.
  • the macromolecules used as reagents and bound to the surfaces of the microporous membrane or which are assayed for using the device in accordance with the present invention generally include materials of a biological nature and are frequently proteinaceous in nature.
  • the reagent or acceptor molecule bound directly to the reaction layer or the ligand being tested for include immunoglobulins or antibodies (either polyclonal or monoclonal), antigenic substances, apoproteins, receptors, glycoproteins, lectins, carbohydrates, hormones, enzymes, carrier proteins, heparin, coagulation factors, enzyme substrates, inhibitors, cofactors, nucleic acids, etc.
  • the diagnostic device or test strip may comprise an elongated strip or support member having sufficient rigidity to allow the test strip to be handled and a test field or indicator medium secured at or near one end of the support member, i.e., near the end opposite that end used as a handle.
  • a second test field or indicator medium may be attached to the side of the support member opposite that to which the test field or indicator medium is attached.
  • test field or indicator medium which is, e.g., approximately one-half the width of the support member, may be secured to a background, untreated medium useful for comparison purposes, i.e., as a baseline or reference.
  • an aperture may be formed near one end of the elongated strip or support member in which the test field or indicator medium is secured. Such a configuration provides ease of liquid penetration and rinsing.
  • a still further embodiment is a test device in which an aperture is formed in the support member in which is secured a composite test field or indicator medium characterized by two or more layers.
  • one layer may be an untreated medium to act as a reference or baseline with the other layer, the treated test field or indicator medium.
  • such a configuration may be read optically by reading first one side, e.g., the reference or baseline side, and then the other side.
  • the elongated support member used in accordance with the subject invention may be made from a variety of materials and take a variety of forms.
  • the material will typically have an elongated, rectangular shape of, for example, 6.4 cm (2.5 inches) to 10.2 cm (4 inches) in length, and 0.5 cm (0.2 inch) to 1.0 cm (0.4 inch) in width.
  • it is a polyester or cellulose acetate film of, for example, 0.2 mm (8/1,000 inch) to 0.4 mm (15/1,000 inch) thick.
  • the criteria for selection of the support member is that it provide sufficient rigidity to allow handling of the test device and that it not interact in any manner with the test liquid, i.e., that it be inert.
  • a device for rapidly and quantitatively detecting the antibody to the bovine hormone glucagon was fabricated in the following manner.
  • IMMUNODYNE TM membrane (a commercially available product of Pall Corporation as described above) having a pore size of 0.2 micrometer and a BET surface area of 8.44 m2/gram was first loaded with glucagon in a controlled fashion as described below.
  • glucagon Product No. 345995, a product of Calbiochem Inc.
  • a 0.1 N aqueous sodium hydroxide solution was dissolved in 1 milliliter of a 0.1 N aqueous sodium hydroxide solution.
  • the membrane was allowed to remain in contact with the solution for one hour to effect chemical binding, after which the membrane was dried in an oven at 40°C for 1/2 hour.
  • the membrane was then immersed in 15 ml of a solution containing 0.5 grams of casein per 100 ml of PBS for 1 hour, washed twice by agitation in successive 25 ml portions of PBS and dried again in an oven at 40°C for 1/2 hour.
  • a 180 mm side roll of 254 micrometers (10 mil) thick cellulose acetate film was coated on one side with a 32 mm wide band of MA-27, an acrylic pressure sensitive adhesive available from Adhesive Research, Inc.
  • the band was applied at a coating weight of 3.2 g/m2 (0.95 oz/yd2) at the center of the film while running lengthwise above the roll as it was unwound.
  • the layer of adhesive was covered with a protective layer of release paper which was siliconized on both sides to effect easy removal or release from the adhesive.
  • IMMUNODYNETM IMMUNODYNETM membrane loaded with glucagon was applied to a 20.3 cm (8 inch) length of the coated cellulose acetate film directly over the 32 mm wide band of adhesive.
  • the IMMUNODYNETM was then pressed against the film with a pressure of about 140 g/cm2 (2 psi) to ensure uniform bonding to the film without damaging the pore structure of the IMMUNODYNETM.
  • the 20.3 cm (8 inch) length of the coated cellulose acetate with the IMMUNODYNETM strip secured thereto was cut into strips 90 mm long and 8 mm wide with the IMMUNODYNETM on one side of one end of the strip and having dimensions of 16 mm (length) by 8 mm (width).
  • Example 1 The device of Example 1 was used to detect and quantify the presence of the antibody to the bovine hormone glucagon in fetal calf serum.
  • the presence of an antibody to one of the animal's own secretions would be suggestive of an autoimmune disease.
  • Typical examples of such diseases are rheumatoid arthritis, and lupus erythematosus.
  • Detection and quantification of antibodies in fetal calf serum are illustrated in Examples 2-5.
  • Example 2 The end of one of the devices prepared as described in Example 1 bearing the membrane indicator medium or test field was immersed in a test tube containing 1.5 ml fetal calf serum for 90 minutes. Upon removal from the serum, the device was washed by agitating it (for 1 minute each time) in three successive 1.5 ml portions of PBS containing 0.1 percent Triton X-100, a non-ionic polyethoxylated nonyl phenol detergent available from the Rohm and Haas Co. It was then agitated (for 1 minute each time) in two successive 1.5 ml portions of PBS.
  • Triton X-100 a non-ionic polyethoxylated nonyl phenol detergent available from the Rohm and Haas Co.
  • the device was thereafter immersed for 90 minutes in 1.5 ml of PBS containing 190,000 counts per minute of 125I-labeled goat anti-rabbit IgG (product number NEX 155 of New England Nuclear). Upon removal from this solution, the device was washed by agitating it (for 1 minute each time) in three successive 1.5 ml portions of PBS containing 0.1 percent Triton X-100. The radioactivity remaining on the device was measured by standard procedures using a LKB/Wallace Mini Gamma gamma ray counter Model No. 1275. The device showed 2019 cpm (counts per minute) of residual gamma activity. These data are tabulated in Table I, as are the data from Examples 3-5 below.
  • Example 2 Another of the devices prepared as described in Example 1 was treated as described in Example 2, except that the fetal calf serum contained 0.004 microliters per milliliter of a commercial preparation of rabbit anti-(bovine glucagon) IgG (product number 969133 available from Calbiochem). When counted for residual radioactivity, the device showed 2302 cpm.
  • Example 3 A third of the devices prepared as described in Example 1 was treated as described in Example 2, except that the fetal calf serum contained 0.04 microliters of the rabbit anti-(bovine glucagon) IgG per milliliter of the fetal calf serum, i.e., the fetal calf serum had a concentration of the antibody ten times (10X) that of the fetal calf serum of Example 3. When counted for residual radioactivity, the device showed 2893 cpm.
  • Example 4 A fourth of the devices prepared as described in Example 1 was treated as described in Example 2, except that the fetal calf serum contained 0.4 microliters of the rabbit anti-(bovine glucagon) IgG per milliliter of the fetal calf serum, i.e., the fetal calf serum had a concentration 100 times (100X) that of the fetal calf serum of Example 3. When counted for residual radioactivity, the device showed 5747 cpm.
  • Example 2 A device was fabricated as described in Example 1, except that the pore size of the IMMUNODYNETM membrane indicator medium or test field used was 5.0 micrometers and the surface area was 3.24 m2/gram. This device was then treated as described in Example 2. When the device was counted for residual radioactivity, it showed 1698 cpm. This data is tabulated in Table II, as is the data from Examples 2 and 3 above and Example 7 below.
  • Example 6 A device was fabricated as described in Example 6. This device was then treated as described in Example 3. When the device was counted for residual radioactivity, it showed 1640 cpm.
  • Table II illustrates the discernible difference in the radioactivity of the devices of Examples 2 and 3, indicating that when the diagnostic device was fabricated using a 0.2 micrometer IMMUNODYNETM indicator medium or test field, the immunoassay was sensitive enough to detect the presence of antibody at a low concentration, i.e., 0.004 ⁇ l/ml. By contrast, when the device was fabricated using a 5.0 micrometer IMMUNODYNETM indicator medium or test field (Examples 6 and 7), no significant difference was detected between the device exposed to an antibody concentration of zero and one exposed to a concentration of 0.004 ⁇ l/ml.
  • an antibody concentration as small as 0.004 ⁇ l/ml could not be detected.
  • a pore size of 0.2 micrometer in the membrane support is preferred over a pore size of 5.0 micrometer because of the increased sensitivity of the resultant assay.
  • the largest reagent of the immunoassay system is of molecular dimensions and is expected to be able to thoroughly penetrate the pore structure of both the 0.2 micrometer and 5.0 micrometer membrane supports.
  • the device in accordance with the present invention can also be used to detect antibodies to whole cells, as well as antibodies to biological molecules.
  • the pores of the membrane support in the device must be large enough to permit penetration of the cells (antigen) into the support. In this manner, maximum sensitivity to the antibody can be achieved.
  • the pore size is too small to permit penetration of the antigen, very small quantities of antibody cannot be detected.

Claims (10)

  1. Diagnostische Vorrichtung zum Testen einer Flüssigkeit für den quantitativen Nachweis der Anwesenheit eines biologisch aktiven Materials in der Flüssigkeit, wobei die Vorrichtung ein längliches Trägerelement aufweist, an dem zumindest in einem Teilbereich eine liquophile, mikroporöse, synthetische, in Alkohol unlösliche Polyamid-Membran oder eine liquophile, mikroporöse, synthetische Polyvinylidendifluorid-Membran als Indikatormedium oder Testfeld befestigt ist, wobei die befestigte Membran eine Oberfläche von 2 bis 12 m²/g, eine Porengröße von 0,04 bis 10 µm mit einer Porengrößenverteilung von nicht mehr als ± 15 % aufweist und die Fähigkeit besitzt, daran ein Reagenz in reproduzierbarer Weise zu immobilisieren.
  2. Diagnostische Vorrichtung nach Anspruch 1, wobei an der Membran eines oder mehrere Reagenzien immobilisiert worden sind.
  3. Diagnostische Vorrichtung nach Anspruch 1, wobei es sich bei der Membran um Nylon 66 handelt.
  4. Diagnostische Vorrichtung nach Anspruch 1, wobei es sich bei der Membran um eine modifizierte Polyamid-Membran mit kontrollierten Oberflächeneigenschaften handelt.
  5. Diagnostische Vorrichtung nach Anspruch 4, wobei es sich bei der Membran um eine Nylon 66-Membran handelt, die mit einem wasserlöslichen Polymer mit einer reichlichen Menge an oberflächlichen Carboxylgruppen modifiziert ist.
  6. Diagnostische Vorrichtung nach Anspruch 5, wobei die modifizierte Membran mit Trichlor-s-triazin behandelt ist.
  7. Diagnostische Vorrichtung nach Anspruch 6, wobei an dem Medium Glucagon immobilisiert ist.
  8. Diagnostische Vorrichtung nach Anspruch 1, wobei die Membran eine Porengröße von 0,1 bis 5 µm aufweist.
  9. Diagnostisches Verfahren zum Testen einer Flüssigkeit zum quantitativen Nachweis eines biologisch aktiven Materials in der Flüssigkeit, umfassend das Kontaktieren der Flüssigkeit mit einem Gehalt an dem zu analysierenden biologisch aktiven Material mit einem liquophilen, mikroporösen, synthetischen Membran-Indikatormedium oder Testfeld mit daran immobilisiertem Reagenz, wobei die Membran ein in Alkohol unlösliches Polyamid oder Polyvinylidendifluorid umfaßt und eine Oberfläche von 2 bis 12 m²/g, eine Porengröße von 0,04 bis 10 µm mit einer Porengrößenverteilung von nicht mehr als ± 15 % aufweist und die Fähigkeit besitzt, das Reagenz daran in reproduzierbarer Weise zu immobilisieren.
  10. Verfahren zur Herstellung einer diagnostischen Vorrichtung für biologisch aktive Materialien, umfassend (1) die Behandlung eines Indikatormediums oder Testfeldmaterials, um daran eines oder mehrere Testreagenzien zu immobilisieren, und (2) die anschließende Herstellung einer diagnostischen Vorrichtung aus einem Teil des behandelten Indikatormediums oder Testfeldmaterials.
EP87311509A 1987-01-12 1987-12-30 Diagnostisches Hilfsmittel, seine Herstellung und seine Verwendung Revoked EP0274911B1 (de)

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US2215 1987-01-12

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EP0274911B1 true EP0274911B1 (de) 1993-03-03

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EP0308704A1 (de) * 1987-09-03 1989-03-29 Fuji Photo Film Co., Ltd. Heterogener Enzym-Immuntest
US5338513A (en) * 1988-07-30 1994-08-16 Boehringer Mannheim Gmbh Test carrier for the analytical determination of a component of a liquid sample
US5075220A (en) * 1988-10-07 1991-12-24 Eastman Kodak Company Determination of a chlamydial or gonococcal antigen using a positively-charged ionically binding support
JPH062142Y2 (ja) * 1988-11-22 1994-01-19 株式会社ニッショー 採尿検査用具
GB2232486A (en) * 1989-05-30 1990-12-12 Quadrant Bioresources Ltd Immunoassay
CA2025474A1 (en) * 1989-09-27 1991-03-28 Donald I. Stimpson Hydrophilic laminated porous membranes and methods of preparing same
CA2025475A1 (en) * 1989-09-27 1991-03-28 Donald I. Stimpson Hydrophilic laminated porous membranes and methods of preparing same
CA2025476A1 (en) * 1989-09-27 1991-03-28 Shan F. Ching Hydrophilic laminated porous membranes and methods of preparing same
WO1991010470A1 (en) * 1990-01-08 1991-07-25 Brown University Research Foundation Devices and methods for enhanced delivery of active factors
DE4015378A1 (de) * 1990-05-14 1991-11-21 Boehringer Mannheim Gmbh Testtraeger fuer die analytische bestimmung eines bestandteils einer probenfluessigkeit
JP2517197B2 (ja) * 1991-02-28 1996-07-24 ベーリンガー・マンハイム・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング 全血中の被分析物測定用テストキャリア
US5116496A (en) * 1991-03-19 1992-05-26 Minnesota Mining And Manufacturing Company Membrane-containing wells for microtitration and microfiltration
GB2257930B (en) * 1991-07-03 1995-04-19 Pall Corp An impervious article with a microporous surface and process for its production
US5344701A (en) * 1992-06-09 1994-09-06 Minnesota Mining And Manufacturing Company Porous supports having azlactone-functional surfaces
US5476665A (en) * 1994-04-13 1995-12-19 Minnesota Mining And Manufacturing Company Azlactone functional particles incorporated in a membrane formed by solvent phase inversion
CA2321100A1 (en) * 1998-02-19 1999-08-26 Edvotek Articles of manufacture and methods for staining biomolecules
CA2462914A1 (en) * 2001-10-11 2003-04-17 Aviva Biosciences Corporation Methods, compositions, and automated systems for separating rare cells from fluid samples

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EP0090483A2 (de) * 1982-02-05 1983-10-05 Pall Corporation Polyamid-Membran und Verfahren zu deren Herstellung
EP0173500A1 (de) * 1984-08-21 1986-03-05 Pall Corporation Methoden zum Konzentrieren von Liganden und dafür verwendete aktive Membranen

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EP0173500A1 (de) * 1984-08-21 1986-03-05 Pall Corporation Methoden zum Konzentrieren von Liganden und dafür verwendete aktive Membranen

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GB2199946A (en) 1988-07-20
GB8730240D0 (en) 1988-02-03
DE3784479D1 (de) 1993-04-08
EP0274911A1 (de) 1988-07-20
DE3784479T2 (de) 1993-06-17
JPS63180857A (ja) 1988-07-25

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